Structural basis of cysteine biosynthesis and peptidoglycan remodelling in Mycobacterium tuberculosis
Author: Böth, Dominic
Date: 2014-01-17
Location: Samuelssonsalen, Tomtebodavägen 6, Scheelelaboratoriet, Karolinska Institutet, Solna Campus
Time: 10.00
Department: Inst för medicinsk biokemi och biofysik / Dept of Medical Biochemistry and Biophysics
Abstract
Mycobacterium tuberculosis is the causative agent of tuberculosis, which is responsible for 1.3 million deaths annually. Treatment is difficult due to an elaborate defense machinery, which includes specific metabolic changes of the bacilli upon exposure to antibiotics and the immune response of the host. The emergence of multi-drug-resistant and extremely drug-resistant strains further complicates the treatment. Therefore the development of novel antibiotics and the investigation of new unexploited targets are of high importance. In the scope of this work, potential drug targets from the L-cysteine biosynthesis and the mycobacterial cell envelope maintenance were investigated.
Cysteine biosynthesis provides promising drug targets due to a direct connection between L-cysteine availability and redox homeostasis of the bacilli. Disruption of the mycobacterial redox defense leads to attenuated growth. Three cysteine synthases are encoded in the mycobacterial genome of which CysK1 and CysM have been characterized previously. In this work CysK2 has been enzymatically characterized, catalyzing the formation of L-cysteine from O-phosphoserine and sulfide in a pyridoxal-5-phosphate-dependent reaction.
The importance of the cell wall integrity makes it a well exploited target of a broad range of antibiotics. Potent inhibitors and first-line antibiotics are currently in clinical use such as isoniazid and ethambutol, which target biosynthetic pathways of the mycobacterial cell wall. RipA, a protein of the NlpC/p60 family was previously validated as essential for infectivity. The characterized NlpC/p60 proteins are peptidoglycan hydrolases and are involved in cell division and peptidoglycan recycling. In M. tuberculosis, four proteins represent this family: RipA, RipB, RipC and RipD. Here we report the biochemical and biophysical characterization of RipA, RipB and RipD as well as their high resolution structures. The detailed understanding of these enzymes prepares the ground for structure-based inhibitor development targeting this enzyme family. RipA and RipB hydrolyze the peptidoglycan peptide stem between D-glutamyl- and meso-diaminopimelic acid residues. RipD is the first NlpC/p60 protein, which adapted to a non-catalytic peptidoglycan-binding function and most likely acts as a scaffold or regulatory protein.
The integrity and stability of the peptidoglycan layer is vital for intracellular survival of M. tuberculosis. L,D- and D,D-transpeptidases strengthen the peptidoglycan layer to withstand chemical and physical stress by the formation of 3-3 and 3-4 cross-links, respectively. The genome of M. tuberculosis encodes five orthologues, the L,D-transpeptidases LdtMt1-5. The three-dimensional structure of LdtMt2 consists of three domains: two smaller domains of the Ig-fold-type segments of the protein (A and B domain) and the transpeptidase domain (C domain). The structure of the two fragments AB domain and BC domain, which are comprising the entire periplasmic part of the enzyme, gives insights to the arrangement of the peptidoglycan layers in the mycobacterial cell wall. Additionally, LdtMt2 has been identified as an off-target for β-lactam antibiotics by the formation of acyl-enzyme complexes with penam and penem class β-lactams.
Cysteine biosynthesis provides promising drug targets due to a direct connection between L-cysteine availability and redox homeostasis of the bacilli. Disruption of the mycobacterial redox defense leads to attenuated growth. Three cysteine synthases are encoded in the mycobacterial genome of which CysK1 and CysM have been characterized previously. In this work CysK2 has been enzymatically characterized, catalyzing the formation of L-cysteine from O-phosphoserine and sulfide in a pyridoxal-5-phosphate-dependent reaction.
The importance of the cell wall integrity makes it a well exploited target of a broad range of antibiotics. Potent inhibitors and first-line antibiotics are currently in clinical use such as isoniazid and ethambutol, which target biosynthetic pathways of the mycobacterial cell wall. RipA, a protein of the NlpC/p60 family was previously validated as essential for infectivity. The characterized NlpC/p60 proteins are peptidoglycan hydrolases and are involved in cell division and peptidoglycan recycling. In M. tuberculosis, four proteins represent this family: RipA, RipB, RipC and RipD. Here we report the biochemical and biophysical characterization of RipA, RipB and RipD as well as their high resolution structures. The detailed understanding of these enzymes prepares the ground for structure-based inhibitor development targeting this enzyme family. RipA and RipB hydrolyze the peptidoglycan peptide stem between D-glutamyl- and meso-diaminopimelic acid residues. RipD is the first NlpC/p60 protein, which adapted to a non-catalytic peptidoglycan-binding function and most likely acts as a scaffold or regulatory protein.
The integrity and stability of the peptidoglycan layer is vital for intracellular survival of M. tuberculosis. L,D- and D,D-transpeptidases strengthen the peptidoglycan layer to withstand chemical and physical stress by the formation of 3-3 and 3-4 cross-links, respectively. The genome of M. tuberculosis encodes five orthologues, the L,D-transpeptidases LdtMt1-5. The three-dimensional structure of LdtMt2 consists of three domains: two smaller domains of the Ig-fold-type segments of the protein (A and B domain) and the transpeptidase domain (C domain). The structure of the two fragments AB domain and BC domain, which are comprising the entire periplasmic part of the enzyme, gives insights to the arrangement of the peptidoglycan layers in the mycobacterial cell wall. Additionally, LdtMt2 has been identified as an off-target for β-lactam antibiotics by the formation of acyl-enzyme complexes with penam and penem class β-lactams.
List of papers:
I. Eva Maria Steiner, Dominic Böth, Robert Schnell and Gunter Schneider. CysK2 (Rv0848): The Third Cysteine Synthase in Mycobacterium tuberculosis. [Manuscript]
II. Dominic Böth, Gunter Schneider and Robert Schnell. Peptidoglycan remodeling in Mycobacterium tuberculosis: Comparison of structures and catalytic activities of RipA and RipB. Journal of Molecular Biology. (2011). 413, 247–60.
Fulltext (DOI)
Pubmed
View record in Web of Science®
III. Dominic Böth, Eva Maria Steiner, Atsushi Izumi, Gunter Schneider and Robert Schnell. RipD (Rv1566c) from Mycobacterium tuberculosis: adaptation of an NlpC/p60 domain to a non-catalytic peptidoglycan-binding function. Biochemical Journal. (2013). 457, 33–41.
Fulltext (DOI)
Pubmed
IV. Dominic Böth, Eva Maria Steiner, Daniela Stadler, Ylva Lindqvist, Robert Schnell and Gunter Schneider. Structure of LdtMt2, an L,D-transpeptidase from Mycobacterium tuberculosis. Acta Crystallographica Section D Biological Crystallography. (2013). D69, 432-41.
Fulltext (DOI)
Pubmed
View record in Web of Science®
I. Eva Maria Steiner, Dominic Böth, Robert Schnell and Gunter Schneider. CysK2 (Rv0848): The Third Cysteine Synthase in Mycobacterium tuberculosis. [Manuscript]
II. Dominic Böth, Gunter Schneider and Robert Schnell. Peptidoglycan remodeling in Mycobacterium tuberculosis: Comparison of structures and catalytic activities of RipA and RipB. Journal of Molecular Biology. (2011). 413, 247–60.
Fulltext (DOI)
Pubmed
View record in Web of Science®
III. Dominic Böth, Eva Maria Steiner, Atsushi Izumi, Gunter Schneider and Robert Schnell. RipD (Rv1566c) from Mycobacterium tuberculosis: adaptation of an NlpC/p60 domain to a non-catalytic peptidoglycan-binding function. Biochemical Journal. (2013). 457, 33–41.
Fulltext (DOI)
Pubmed
IV. Dominic Böth, Eva Maria Steiner, Daniela Stadler, Ylva Lindqvist, Robert Schnell and Gunter Schneider. Structure of LdtMt2, an L,D-transpeptidase from Mycobacterium tuberculosis. Acta Crystallographica Section D Biological Crystallography. (2013). D69, 432-41.
Fulltext (DOI)
Pubmed
View record in Web of Science®
Institution: Karolinska Institutet
Supervisor: Schneider, Gunter
Issue date: 2013-12-12
Rights:
Publication year: 2013
ISBN: 978-91-7549-388-6
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